We are pursuing an HIV vaccine approach based on replication-competent Adenovirus (Ad)-recombinants. The rationale for this strategy is based on the fact that live attenuated vaccines historically have been the most protective, eliciting essentially life-long immunity. Examples include vaccines for small pox, polio, measles, and yellow fever. We conduct pre-clinical vaccine studies in rhesus macaques and challenge with SIV or SHIV (a chimeric SIV virus containing an HIV envelope), systems that model HIV-infection of humans. We use a prime-boost strategy, first immunizing with a replicating adenovirus (Ad) vector carrying an HIV/SIV gene(s) followed by a boosting with HIV/SIV envelope protein. Ad replicates in epithelial cells that line mucosal inductive sites, and therefore elicits strong, persistent cellular immunity at mucosal effector sites as well as in the blood. We recently conducted a pre-clinical vaccine study in rhesus macaques and investigated cellular immune responses elicited by the vaccine regimen. Of note, while cellular immunity did not influence SIV acquisition, vaccine-induced CD8 T cells not unexpectedly correlated with decreased viremia. This correlation was also observed in male macaques, of particular interest since mucosal immunity and B cell responses were associated with delayed SIV acquisition in females. An on-going pre-clinical study is evaluating if these protective responses observed in males and females are reproduced. Gamma-delta T cells, effector cells that are especially important with regard to mucosal immune protection, have also been investigated in these preclinical studies, which have focused in particular on the female reproductive tract, as well as rectal tissue and blood. We have characterized the various cell populations phenotypically and functionally, and shown that gamma-delta T cells in the ectocervix and endocervix, as well as in the circulating population, contribute to control of viremia. These observations, especially regarding the female reproductive tract, are novel and applicable not only to control of HIV/SIV infection, but most likely to mucosal pathogens in general. We have also studied effector cells which mediate non-neutralizing antibody responses which have been shown to contribute to protective efficacy. Natural killer (NK) cells are a key population which can kill target cells directly and also mediate antibody-dependent effector functions such as antibody-dependent cellular cytotoxicity (ADCC). A comparison of NK cells in uninfected and chronically infected macaques revealed that the natural cytotoxic capacity of the cells was lost with SIV infection, and a greater concentration of IL-15 was necessary to restore this capability. In contrast the ability to mediate Fc effector functions was not impaired. A current study is exploring whether memory NK cells exist in non-human primates (as has been shown in mice) and whether vaccination impacts this cell subset. Additionally, the impact of NK cells in mucosal tissues is under investigation. T follicular cytotoxic T cells have been recently identified in lymph nodes. Our studies are showing that this cell population is associated with viremia control, and is dependent on interactions with both T follicular helper cells and T follicular regulatory cells. Impaired Tfc responses are observed in SIV-infected macaques that fail to control viremia. Finally, phenotypic and functional studies of macrophages and dendritic cells over the course of vaccination of non-human primates and subsequent challenge with infectious SIV are underway. Our vaccine strategy using replicating adenovirus recombinants targets mucosal inductive sites, particularly in the upper respiratory tract, where macrophages are especially prevalent. The vector also has a broad biodistribution, and has been shown to persist in rectal mucosal tissue. The functional activities of macrophages in the lung and cervical mucosa are now being investigated in depth. Dendritic cells are critical for induction of adaptive immune responses, and are similarly being studied over the course of immunization and viral challenge. The role they play in the adenovirus-based vaccine strategy will inform improved design of vaccine immunogens. Overall our studies of cells of both the innate and adaptive immune systems will provide valuable information able to advance the goal of achieving a protective HIV vaccine.